Modern research methods for determining structures of intradiol dioxygenases Danuta WOJCIESZYŃSKA, Katarzyna HUPERT-KOCUREK, Małgorzata SITNIK, Urszula GUZIK – Department of Biotechnology, Faculty of Biology and Environmental Protection, University of Silesia in Katowice Please cite as: CHEMIK 2012, 66, 12, 1346-1351 science • technique Introduction β subunits, near the peak of the symmetry axis, there is an active site. The In the era of a large interest in biodegradation processes of subunits are homologous towards each other. β subunit is composed aromatic compounds, considerable attention has been directed of ca. 200 amino acid residues, while α subunit is composed of 230 to the studies on degrading enzymes, among which aromatic ring- amino acid residues. The secondary structure has the conformation cleavage dioxygenases have a key meaning [1,2]. Such enzymes of β-barrel constituted of β-sheet structure that is composed of catalyse the opening of a ring as a result of attaching two atoms of 8 chains, twisted and coiled into the closed structure. It is similar to molecular oxygen and they are classified into two following groups a piece of paper when folded in half [2, 5, 6]. depending on the regiospecificity of the ring cleavage: intradiol and Catechol 1,2-dioxygenases belong to the widely described class of extradiol [3]. The ring cleavage with the participation of intradiol enzymes [EC 1.13.11.1]. These enzymes cleave the catechol aromatic dioxygenases does not only provide the degradation of uneasily ring into cis,cis-muconic acid. Regarding the catalysed substrate, two degradable aromatic compounds such as: chlorophenols and subclasses of these enzymes are distinguished. They are: nitrophenols, biphenyls and polycyclic aromatic hydrocarbons, but I – catechol dioxygenases catalysing the decomposition of catechol it also results in obtaining valuable intermediates for organic syntheses and methylcatechol, and less often chlorocatechol (cis, cis -muconic acid and 3-carboxy- cis, cis -muconic acid). II – chlorocatechol dioxygenases decomposing catechol as well as its chlorinated and methylated forms [7÷10]. Intradiol dioxygenases – enzymes involved in decomposition Catechol dioxygenases are homodimers of two identical of aromatic compounds α subunits, both of which contain Fe(III) cofactor. Each subunit is Intradiol dioxygenases catalyse the opening of the aromatic ring composed of ca. 300 amino acids and coiled into two domains: between two hydroxylated carbon atoms of the aromatic system, the catalytic domain whose structure is similar to the core of initiating the decomposition of aromatic compounds in the ortho protocatechuate dioxygenases, and the terminal domain involved pathway. It is a small family of enzymes with nonheme iron (III). in dimerisation. N-terminal domain consists of ca. 100 amino acid They probably originate from the same evolutionary line. The residues making 5 helices. Dioxygenases of this class differ from analysis of spatial structure and amino acid sequence formed the other intradiol dioxygenases regarding both the subunit structure and basis for dividing dioxygenases from this group into the following: the helical connector present at the subunit boundaries, to which catechol 1,2-dioxygenases composed of α subunits, protocatechuate phospholipid is being attached. The role of phospholipids has not 3,4-dioxygenases composed of different numbers of αβ subunits and been closely investigated so far [5, 6, 9]. hydroxyquinol 1,2-dioxygenases composed of α subunits, similarly Hydroxyquinol 1,2-dioxygenases belong to 3 rd group of intradiol to catechol dioxygenases. Despite different subunit composition, dioxygenases [EC 1.13.11.37]. They catalyse hydroxyquinol catalytic domains of intradiol dioxygenases are formed similarly. transformation into 3-hydroxy- cis, cis -muconate. The model of In the active site of intradiol dioxygenases, there is iron in trigonal spatial structure was described for hydroxyquinol dioxygenase of bipyramidal coordination geometry bound with four endogenous Nocardioides simplex strain 3E. It is a homodimer having the dimensions protein ligands. In the trigonal bipyramidal formation, the central of 110x50x50 Å. Its structure and subunit composition ( α2) are similar atom is linked to 5 molecules at the tops of both pyramids [2]. to catechol dioxygenase, which is related to their close affinity. Tyrosine 408, Histidine 460 and hydroxy group are attached to Fe (III) Despite that close affinity with catechol dioxygenases, hydroxyquinol in the equatorial plane, whereas Tyrosine 447 and Histidine 462 in the dioxygenases have specific amino acid residues in their structure axial plane. The composition of the active site is closely related to its (Leu80, Asp83, Val107, Phe108, Gly109, Pro110, Phe111,Ile199, function. The tests on the structure of enzyme-substrate complex Pro200, Arg218, Val251) and big openings for attaching the substrate of protocatechuate 3,4-dioxygenase showed that the attachment of in the upper part of the active site [5, 8]. substrate was associated with the separation of hydroxy group and The intradiol mechanism of ring cleavage has not been fully Tyrosine 447, and the attached substrate gave its two protons to the investigated. The mechanism of intradiol aromatic ring cleavage detached ligands [4, 5]. was suggested on the basis of the analysis of the spatial structure of Protocatechuate 3,4-dioxygenase is the enzyme that catalyses the protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase and transformation of protocatechuic acid into 3-carboxy-cis, cis-muconic enzyme-substrate complexes as well as on the basis of biochemical acid [1,6,7]. This enzyme is characterised by the oligomer structure and spectroscopic properties. and is composed of two different types of subunits – α and β forming The process of ring cleavage began from substrate attachment the (αβFe)n structure. β subunits combine protomers forming the to the active site. The substrate gave two protons – one to hydroxy oligomer structure and they are arranged along the axis of tetrahedron group, and the second to tyrosine in the axial position, which caused the symmetry making the hollow sheath having the diameter of 50Å. deprotonation of both hydroxy groups of the substrate. Simultaneously, α subunits are arranged in proximity to the peaks of two axes; they the dissociation of ligands coordinated with iron from the active site and can be also found in the corners of opposite walls. Between α and the attachment of the dianonic substrate were observed. This led to the nr 12/2012 • tom 66 • 1349 formation of chelate ring activating the electrophilic attack of oxygen spholipid chains, whereas the regions characterised by a weaker si - molecule. The process outcome was the formation of peroxy bond gnal could suggest the presence of hydrophilic part of phospholipid. between iron and substrate. Tyrosine in the equatorial arrangement The signals obtained for the standard phospholipids were compared facilitated ketonization between C3-O, which resulted in arginine- in order to determine the types of phospholipids present in the he - stabilised carbanion. Arginine along with tyrosine controlled the direct lical connector of the tested enzyme. It was the basis for identifying attack of oxygen molecule. Consequently, this resulted in opening of two following phospholipids: glycerophosphoinositol and glycero - the ring structure and releasing the product – cis, cis-muconic acid or phosphoinositol monophosphate. However, an attempt to deter - its derivative [2, 11, 12]. mine unambiguously which of them was present in the connector failed due to too weak signal in the regions of hydrophilic part of Research methods used to determine structures of intradiol the tested phospholipid [9]. dioxygenases X-ray absorption (XAS) spectroscopy is a widely applied technique The research works on intradiol dioxygenase properties, belonging to atomic absorption spectrometry. It employs the action including their structures, have been a subject of interest for a few of X-ray radiation on a tested object. XAS was used to test the active science • technique years. Nowadays, the studies on the enzymes widely apply the sites of hydroxyquinol 1,2-dioxygenase of N. Simplex strain 3E and of methods related to two following fields: crystallography (X-ray chlorocatechol 1,2-dioxygenase of R. erythropolis strain 1CP. Native crystallography) and spectroscopy (mass spectrometry, electron enzymes and their complexes with substrates were compared. The paramagnetic resonance (EPR) spectroscopy and X-ray absorption obtained spectra were typical for the metal combined with oxygen (XAS) spectroscopy). and nitrogen ligands. The performed calculations suggested the At first, crystallography was related to mineralogy and materials presence of Fe(III) as chromophore coordinated with 5 ligands. The science. At present, it is applied in many branches of industry and noticeable weaker signal and interference during the formation of science, including biology, since it allows the ordered structure of enzyme-substrate complex were also important. They indicated the a molecule, such as e.g. proteins, to be presented [13]. The X-ray significant change in the active site resulting from the direct interaction crystallography has been applied for determining the dimensions of substrate with metal ion in the enzyme active site. On the basis and geometry of crystalline lattice formed by a unit cell. The of the observation, it was assumed that the substrates